There has been an intense interest in the structure and function of the regulatory regions controlling the expression of genes in the pathway for methanol utilization. The methylotrophic yeasts are found in four genera that can be divided into two groups the ascosporogenous Hansenula and Pichia, and the asporogenous Candida and Torulopsis. The first enzyme in the methanol-utilization pathway is alcohol oxidase. It catalyzes the oxidation of methanol to formaldehyde. During this reaction there is a simultaneous reduction of oxygen to hydrogen peroxide. When glucose grown yeast cells are transferred to methanol containing media as a sole carbon source, the peroxisome, a subcellular organelle, begins to swell and proliferate. Large amounts of alcohol oxidase are sequestered into the peroxisomes. The compartmentalization of alcohol oxidase in the peroxisomes protects the cytosol from hydrogen peroxide. The high concentration of this enzyme compensates for its low affinity for oxygen. In methanol-grown Pichia pastoris cells, alcohol oxidase constitutes up to 30% of the total soluble protein.
Alcohol oxidases have numerous commercial applications. These uses include the measurement of alcohol levels in various biological and nonbiological fluids, and the conversion of alcohol precursor molecules into aldehydes, e.g., for use in artificial flavor production.
Several genes encoding alcohol oxidase from Pichia pastoris (AOX1, AOX2), Candida boidinii S2 (AOD1), and methanol oxidase from Hansenula polymorpha (MOX1) have been isolated and characterized. The regulation of the synthesis of alcohol oxidase is primarily controlled at the level of transcription. During methanol induction, the rapid de novo synthesis of the enzyme is accompanied by a dramatic increase in alcohol oxidase mRNA. Previous studies by Ellis et al., Mol. Cell. Biol, 5: 1111-1121 (1985) of the AOX1 promoter indicates that it is strongly repressed by ethanol and glucose. Based on northern hybridizations, Pichia pastoris cells grown in the presence of ethanol did not synthesize alcohol oxidase specific poly(A).sup.+ RNA. The synthesis of alcohol oxidase is tightly catabolite repressed. In cells grown in glucose containing media, alcohol oxidase mRNA is not detectable. In order to study the regulation of the AOX1 gene Tschopp et al., Nucl. Acids Res., 15: 3859-3876 (1987) took the AOX1 promoter and fused it to the E. coli lacZ gene. Saccharomyces cerevisiae strains harboring an AOX1-lacZ fusion produced only small amounts of active enzyme when grown in glucose or ethanol. See Stroman et al., U.S. Pat. No. 4,855,231.
The methylotrophic yeasts Hansenula polymorpha and Pichia pastoris have been used as hosts for heterologous gene expression. Cregg et al., Bio/Technology, 5: 479-485 (1987); Tschopp et al., Bio/Technology, 5: 1305-1308 (1987). Over twenty proteins of potential commercial value have been produced using methanol regulated promoters. These heterologous proteins can accumulate to high levels in the cytoplasm (tumor necrosis factor was expressed at 8 g/liter, Skeekrishna et al., Biochemistry, 28: 4117-4125, 1989) or can be secreted into the media (Saccharomyces cerevisiae invertase was secreted at 2.5 g/liter, Tschopp et al., Bio/Technology, 5: 1305-1308, 1987). Stable transformants have been obtained by integrating the alcohol oxidase promoter expression cassette into the yeast chromosomes. Increases in the level of expression have been obtained by increasing the number of integrated copies of the heterologous gene expression cassette (tetanus toxin fragment C was expressed at 12 g/liter, Clare et al., Bio/Technology, 9: 455-460).
Methylotrophic yeasts have been used in the production of heterologous proteins. Increases in gene dosage, cell density, and promoter strength have resulted in high-level expression of valuable proteins. Several genes encoding alcohol oxidase (Pichia pastoris AOX1, AOX2, and Candida Boidinii S2 AOD1), methanol oxidase (Hansenula polymorpha MOX), and dihydroxyacetone synthase (Hansenula polymorpha DHAS) have been isolated and characterized, see, for example, Ellis et al., Mol. Cell. Biol, 5: 1111-1121 (1985) (AOX1); Koutz et al., Yeast, 5: 167-177 (1989) (AOX2); Sakai et al., Gene, 114: 67-73 (1992) (AOD1); Ledeboer et al., Nucl. Acids Res., 13: 3063-3082 (1985) (MOX1). The synthesis of these enzymes is tightly controlled by methanol induction and glucose catabolite repression. When Pichia pastoris is grown on methanol as a sole carbon source, alcohol oxidase (a peroxisomal packaged enzyme) constitutes up to 30% of the total soluble protein.
Although high-level expression of heterologous proteins has been achieved using some methylotrophic yeast promoters, very little is known about the molecular mechanism involved in methanol induction. Previous studies comparing the methanol regulated promoters did not reveal any significant regions of homology. Koutz et al., Yeast, 5: 167-177 (1989); Sakai et al., Gene, 114: 67-73 (1992); Ledeboer et al., Nucl. Acids Res., 13: 3063-3082 (1985). A possible mechanism for the methanol induction of the methanol regulated gene is by means of a positive effector molecule that activates transcription by binding at specific DNA sequences, e.g., gene regulatory proteins. Thus, it is of interest to identify nucleotide sequences that are conserved among methanol regulated genes so as to identify molecules involved in their expression and regulation, and to confer similar forms of regulation on heterologous genes.
Given the recognized utility of methylotrophic yeast, as well as other yeast, in the expression of heterologous proteins, it is of interest to provide promoters and other regulatory nucleotide sequences for the controlled and/or high level expression of heterologous proteins in yeast. It is also of interest to identify new methanol regulated genes so as to provide for the increased production of the proteins encoded by these methanol regulated genes and to provide for nucleotide sequences involved in the expression of methanol regulated genes.
The breakdown of starch is an important process in the brewing, baking, and sweetener industries. The degradation of the starchy cereal grain endosperm is initiated by .alpha.-amylase. In rice (Oryza sativa), .alpha.-amylase is a monomeric Ca.sub.2+ -requiring metalloprotein which catalyzes an .alpha.(1-4) endoglycolytic cleavage of amylose and amylopectin. Since the yeast Saccharomyces cerevisiae which is commonly used in industry lacks the genes encoding for .alpha.-amylase, it is unable to directly ferment starch. During the commercial production of alcoholic beverages the starch is initially pretreated with an exogenous source of .alpha.-amylase.
Although the expression of a wheat .alpha.-amylase in yeast has been reported previously (Rothstein et al., Gene, 55: 353-356, 1987), the secretion of this protein across the cell membrane was minimal. Because the wheat a-amylase gene used is their study did not encode an enzyme containing an N-glycosylation site, the low level of secretion may by due to the lack of N-glycosylation. The secretion of barley .alpha.-amylase in yeast had higher levels of expression (Sogaard et al., Gene, 94: 173-179, 1990). The highest yield was 2-3 .mu.g/ml of barley .alpha.-amylase AMY1 under the control of the 3'-phosphoglycerate kinase promoter. It is interesting to note that the rice .alpha.-amylase is the only cereal amylase known to be glycosylated. Kumagai, et al., Gene, 94: 209-216 (1990) expressed .alpha.-amylase OS103 (O'Neill et al., Mol. Gen. Genet., 221: 235-244, 1990) in a laboratory strain of Saccharomyces cerevisiae under the transcriptional control of the enolase promoter. In that study, during the biosynthesis of rice .alpha.-amylase by yeast, the plant signal peptide was removed, the protein was N-glycosylated, and the active, enzyme was secreted into the culture media. Since the laboratory strain of Saccharomyces cerevisiae (LL20) used in that study was deficient in the enzyme maltase the recombinant strain LL20 [pEno/1031 is unable to produce significant amounts of ethanol. The reported yield of secreted enzyme was 1.8 .mu.g/ml of rice .alpha.-amylase OS103.
Other amylolytic genes have been expressed in Saccharomyces cerevisiae cDNAs encoding mouse (Thomsen, Carlsberg Res. Commun., 48: 545-555, 1983; Filho et al., Bio/Technology, 4: 311-315, 1986) and human salivary gland .alpha.-amylase (Sato et al., Gene, 50: 245-247, 1986), and Aspergillus awamori (Innis et al., Science, 228: 21-26, 1985) are some of the previous examples. Inlow et al., Biotechnology and Bioengineering, 32: 227-234, (1988) were able to convert Maltrin 150 (Grain Processing Corporation, Mucatin, IA) to ethanol. Maltrin 150 is a soluble starch which is enzymatically hydrolyzed by .alpha.-amylase.
Alcohol oxidases have numerous commercial applications. These uses include the measurement of alcohol levels in various biological and non-biological fluids, and the conversion of alcohol precursor molecules into aldehydes, e.g., for use in artificial flavor production.
Previous studies by Ellis et al., Mol. Cell. Biol, 5: 1111-1121 (1985) of the AOX1 promoter indicates that it is strongly repressed by ethanol and glucose. Based on Northern hybridizations, Pichia pastoris cells grown in the presence of ethanol did not synthesize alcohol oxidase specific poly(A).sup.+ RNA. In order to study the regulation of the AOX1 gene Tschopp et al., Nucl. Acids Res., 15: 3859-3876 (1987) took the AOX1 promoter and fused it to the E. coli lacZ gene. Saccharomyces cerevisiae strains harboring an AOX1-lacZ fusion produced only small amounts of active enzyme when grown in glucose or ethanol. See Stroman et al., U.S. Pat. No. 4,855,231. This result indicates that the AOX1 promoter would not be useful in the production of heterologous proteins during ethanol production in Saccharomyces cerevisiae.
The subject invention provides for alcohol oxidase promoters that can direct high level expression of rice .alpha.-amylase in the brewer's yeast Saccharomyces cerevisiae and in other yeast species. The ZZA1 regulatory region contains several nucleotide sequences involved in promoter activity. These sequences include a TATAA box, located 45 bp upstream of the putative transcription initiation site. In order to find additional regulatory sites, the nucleotide sequence of ZZA1 promoter was compared to AOX1, AOX2, MOX1, AOD1, and DHAS genes. The highly conserved regions between these genes may be involved in binding methanol specific transacting factors. A region in the core consensus sequence of ZZA1 (GCTTCCA, position -123) is identical to the sequence which is adjacent the S. cerevisiae regulatory activating protein (RAPL) binding site, as noted in Buchanan et al., Mol. Cell. Biol., 8: 210225, Brindle et al., Mol. Cell. Biol., 10: 4872-4885. Deletion of the terminal G of the GCTTCCA sequence causes a loss in ENO1 gene expression. The CTTCC motif may also serve as a binding site for the trans-acting factor GCR1. This regulatory protein is required for high-level expression of several glycolytic genes in S. cerevisiae Huie, et al. Mol. Cell. Biol., 12: 26900-2700 (1992).